This project, which has been funded for over 30 years, addresses fundamental mechanisms of insulin action on adipose metabolism and functions that control whole body metabolic homeostasis in health and disease. Co-morbidities associated with obesity including insulin resistance and type 2 diabetes are thought to result from insufficient deposition and storage of triglyceride within adipose tissue, leading to deleterious effects of fat on liver, skeletal muscle, cardiovascular organs and insulin secreting beta cells. This project previously employed siRNA screens to discover the Ste20 ortholog Map4k4, a protein kinase that acts as a critical signaling node in many tissues related to metabolic homeostasis. In the previous grant period we have carefully assessed the role of Map4k4 in vivo showing that this protein kinase functions in many tissues to suppress systemic insulin sensitivity as well as promote atherosclerosis. Thus, we consistently observed beneficial metabolic phenotypes in six tissue- specific Map4k4 knockout mouse models we produced, demonstrating that Map4k4 exerts powerful biological effects worthy of continued study. Highlights of these studies include: 1. endothelial cell Map4k4 KO lowers blood glucose and attenuates atherosclerosis in ApoE null mice, 2. Inducible whole body Map4k4 KO mice show increased insulin sensitivity, much lower insulin levels and compromised insulin secretion from islets, 3. Map4k4 KO in Myf5 positive cells (but not in Adiponectin positive cells) causes a marked increase in adipocyte insulin sensitivity and Akt activation. This latter finding is consistent with the exciting hypothesis that skeletal muscle secretes one or more factors that enhance insulin sensitivity of adipocytes. Overall, our findings also suggest that two protein kinases very highly similar to Map4k4, Tnik and Mink, may also be active in the same pathways. We have therefore produced triple floxed Tnik/Mink/Map4k4 mice in order to test their potential redundancy with Map4k4 function in adipocytes (lipid metabolism) by crossing with adiponectin-Cre mice and in beta cells (insulin secretion) by crossing with Ins1Cre or Ins1CreER mice (Aim 1). We also propose to rigorously test the exciting hypothesis that Map4k4 KO in Myf5 positive cells induces a circulating factor that enhances adipocyte insulin sensitivity and glucose tolerance by parabiosis studies with the mouse models we have engineered (Aim 2). Finally, in parallel we seek to identify such circulating factors in serum using Somalogic technology and mass spectroscopy in the UMASS Proteomics Core Facility (Aim 3). RELEVANCE (See instructions): Fat tissue plays a key role in regulating energy metabolism, and excess accumulation of fat in obesity is associated with many maladies including type 2 diabetes. We identified components of signaling pathways in fat cells that mediate dysfunction in obesity and have studied an enzyme called Map4k4 that contributes to insulin resistance, atherosclerosis and other diseases associated with metabolic functions. We hope to further characterize Map4k4's functions, as it may be a good target for new drug therapies.